The objectives of this research are to study the (i) organization and regulation of genes encoding glutamine amidotransferase enzymes, (ii) relationships of structure to glutamine amide transfer function and mechanisms for catalysis in enzymes having two different glutamine amide transfer domains, and (iii) the role(s) of the (4Fe-4S) centers in an amidotransferase and a second, well characterized enzyme, aconitase. The experimental approach will emphasize molecular biology. The cloned genes to be used include: bacterial trpEG (anthranilate synthase), E. coli pyrG (CTP synthetase), E. coli and B. subtilis purF (amidophosphoribosyltransferase), and a large cluster of pur genes from B. subtilis. In addition, pig heart aconitase cDNA will be cloned. The specific aims are: (i) Employ sequence comparisons of homologous glutamine amide transfer domains to infer conserved, possibly essential amino acid residues, sitedirected mutagenesis to replace inferred essential residues, and techniqus of enzyme chemistry to determine the role of functional residues in catalysis. This same approach will be used to investigate allosteric regulatory sites in three amidotransferases. (ii) The cloning and nucleotide sequence of a cluster of at least 12 genes involved in purine nucleotide synthesis in B. subtilis will be completed. This cluster likely contains all of the genes for the de novo pathway to IMP. The expression and unique regulation of the gene cluster will be studied. (iii) In vitro and in vivo mutational analyses will be used to study the promoter and regulatory region of E. coli purF. A plan is described to isolate an unlinked regulatory mutation which will then be used to clone the putative trans-acting regulatory gene. (iv) The maturation of B. subtilis amidophosphoribosyltransferase, which involves NH2-terminal undecapeptide clipping and assembly of a (4Fe-4S) center, will be studied in bacterial and CHO cells. Site-directed mutations are planned to determine whether the undecapeptide leader has a role in the assembly of the (4Fe-4S) center, and whether processing of the leader requires factors or is autocatalytic. This B. subtilis enzyme is a good model for the presently unavailable human enzyme, which may have a role in gout. (v) Cloning and sequence analysis of aconitase cDNA will initiate studies on a second (4Fe- 4S) enzyme and will complement the X-ray structural analysis. Inhibition of tumor cell growth by drugs such as acivicin, a specific inhibitor of glutamine amidotransferases, illustrates the essential role of this group of enzymes in cell growth and the importance of these enzymes.